Nucleic Acid Modification—Polynucleotides can be covalently modified using a large number of different methodologies. Direct covalent attachment of molecules to polynucleotides can be accomplished by nitrogen mustards (alkylation), 2-acetylfluorene, 4-aminohydroxylamine, p-diazobenzoyl-biocytin, bisulfite activation, n-bromosuccinimide activation, EDC modification of 5′ phosphates and with photobiotin (N-(4-azido-2-nitrophenyl)-aminopropyl-N′-(N-d-biotinyl-3-aminopropyl)-N′-methyl-1,3-propanediamine. Polynucleotides (DNA or RNA) can also by synthesized via in vitro enzymatic reactions to include covalently modified nucleotides. These modified nucleotides, which are incorporated into the growing polynucleotide chain, can be chemically coupled to a wide array of molecules. Some examples of molecules that can be covalently linked to polynucleotides directly or through enzymatic incorporation of modified nucleotides include; fluorescent molecules (fluorescein, rhodamine, cyanine dyes, ALEXA™ dyes), peptides (i.e. nuclear localizing signals), proteins (enzymes, ligands, antibodies), lipids, sugars, carbohydrates, biotin, avidin, streptavidin, chemiluminescent substrates, digoxin and dinitrophenyl (DNP). Thus using any of these compounds or methods a vast array of molecules or compounds can be covalently attached to polynucleotides.
Nucleic Acid Alkylation—Nucleic acid alkylation results in the formation of a chemical bond between the alkylating compound (labeling reagent) and the polynucleic acid. In an alkylation reaction the polynucleic acid is incubated with the said compounds in aqueous or non-aqueous solutions, followed by separation of the labeled polynucleic acid from the unreacted alkylating reagent. The extent of alkylation can be controlled by regulating the relative amounts of alkylating reagent and polynucleic acid, by adjusting the length of the incubation, by controlling the temperature of the incubation, by controlling the absolute concentrations of polynucleic acid and alkylating reagent, and by controlling the composition of the aqueous or organic solution using solvent, pH, ionic strength, and buffers.
Condensation of DNA—A large number of multivalent cations with widely different molecular structures have been shown to induce change in the tertiary structure of DNA including condensation.
Two approaches are currently used for compacting (condensing) DNA:                1. Multivalent cations with a charge of three or higher have been shown to condense DNA. These include spermidine, spermine, Co(NH3)63+, Fe3+, and natural or synthetic polymers such as histone H1, protamine, polylysine, and polyethylenimine. Analysis has shown DNA condensation to be favored when 90% or more of the charges along the sugar-phosphate backbone are neutralized by the polycation.        2. Polymers (neutral or anionic) which can increase repulsion between DNA and its surroundings have been shown to compact DNA. Most significantly, spontaneous DNA self-assembly and aggregation process have been shown to result from the confinement of large amounts of DNA, due to excluded volume effect.        
Depending upon the concentration of DNA, condensation leads to three main types of structures:                1. In extremely dilute solution (about 1 μg/ml or below), long DNA molecules can undergo a monomolecular collapse and form structures described as toroids.        2. In very dilute solution (about 10 μg/ml) microaggregates form with short or long molecules and remain in suspension. Toroids, rods and small aggregates can be seen in such solution.        3. In dilute solution (about 1 mg/ml) large aggregates are formed that sediment readily.        
Toroids have been considered an attractive form for gene delivery because they have the smallest size. While the size of DNA toroids produced within single preparations has been shown to vary considerably, toroid size is unaffected by the length of DNA being condensed. DNA molecules from 400 bp to genomic length produce toroids comparable in size. Therefore one toroid can include from one to several DNA molecules. The kinetics of DNA collapse by polycations that resulted in toroids is very slow. For example DNA condensation by Co(NH3)6Cl3 needs 2 hours at room temperature.
The mechanism of DNA condensation is not obvious. The electrostatic force between unperturbed helices arises primarily from a counterion fluctuation mechanism requiring multivalent cations and plays a major role in DNA condensation. The hydration forces predominate over electrostatic forces when the DNA helices approach closer then a few water diameters. In the case of DNA—polymeric polycation interactions, DNA condensation is a more complicated process than the case of low molecular weight polycations. Different polycationic proteins can generate toroid and rod formation with different size DNA at a ratio of positive to negative charge of 0.4. T4 DNA complexes with polyarginine or histone can form two types of structures; an elongated structure with a long axis length of about 350 nm (like free DNA) and dense spherical particles. Both forms exist simultaneously in the same solution. The reason for the co-existence of the two forms can be explained as an uneven distribution of the polycation chains among the DNA molecules. The uneven distribution generates two thermodynamically favorable conformations. The electrophoretic mobility of DNA-polycation complexes changes from negative to positive when there is an excess of polycation. This results in DNA condensation and particle formation and the DNA-polycation complexes remain in the well during electrophoresis In the absence of an excess of a polycation or oligocation, the DNA remains unaggregated and the DNA and polycations can dissociate allowing the DNA to migrate into an agarose gel during electrophoresis. In DNA/polycation complexes (not covalently or chemically attached) it is likely that the large polycations don't completely align along DNA but form polymer loops that interact with other DNA molecules. The rapid aggregation and strong intermolecular forces between different DNA molecules may prevent the slow adjustment between helices needed to form tightly packed orderly particles.